Trypanocidal activity and selectivity in vitro of aromatic amidine compounds upon bloodstream and intracellular forms of Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas disease, an important neglected illness affecting about 12-14 million people in endemic areas of Latin America. The chemotherapy of Chagas disease is quite unsatisfactory mainly due to its poor efficacy especially during the later chronic phase and the considerable well-known side effects. These facts emphasize the need to search for find new drugs. Diamidines and related compounds are minor groove binders of DNA at AT-rich sites and present excellent anti-trypanosomal activity. In the present study, six novel aromatic amidine compounds (arylimidamides and diamidines) were tested in vitro to determine activity against the infective and intracellular stages of T. cruzi, which are responsible for sustaining the infection in the mammalian hosts. In addition, their selectivity and toxicity towards primary cultures of cardiomyocyte were evaluated since these cells represent important targets of infection and inflammation in vivo. The aromatic amidines were active against T. cruzi in vitro, the arylimidamide DB1470 was the most effective compound presenting a submicromolar LD(50) values, good selectivity index, and good activity at 4 °C in the presence of blood constituents. Our results further justify trypanocidal screening assays with these classes of compounds both in vitro and in vivo in experimental models of T. cruzi infection.

Guidelines for protein design: the energetics of beta sheet side chain interactions.

To determine the interaction energy between cross-strand pairs of side chains on an antiparallel beta sheet, pairwise amino acid substitutions were made on the solvent-exposed face of the B1 domain of streptococcal protein G. The measured interaction energies were substantial (1.8 kilocalories per mole) and comparable to the magnitude of the beta sheet propensities. The experimental results paralleled the statistical frequency with which the residue pairs are found in beta sheets of known structure.

Expression of bovine myf5 induces ectopic skeletal muscle formation in transgenic mice.

myf5 is one of a family of four myogenic determination genes that control skeletal muscle differentiation. To study the role of myf5 in vivo, we generated transgenic mice harboring the bovine homolog, bmyf, under control of the murine sarcoma virus promoter. Ectopic expression of the full-length bmyf transgene was detected in brain and heart tissue samples of F1 progeny from transgenic founder mice. Ectopic bmyf expression activated endogenous skeletal myogenic determination genes in the hearts and brains of transgenic animals. Incomplete skeletal myogenesis in most hearts gave rise to cardiomegaly and focal areas of cardiomyopathy. In brains in which ectopic expression led to a more complete myogenesis, focal areas of multinucleated, striated myotubes containing actin, desmin, and myosin were observed. These unexpected results show that myf5 can initiate myogenic differentiation in vivo, supporting the hypothesis that myf5 is responsible for determination of cells to the myogenic lineage in normal embryogenesis.

The influence of the fatty acid composition of Acholeplasma laidlawii membranes on the growth inhibitory activity of narasin, a polyether ionophorous antibiotic.

Narasin, apolyether ionophorous antibiotic capable of acting as a transmembrane carrier of cations, has a growth inhibitory effect on Acholeplasma laidlawii, permitting only 20% survival when present as 0.1 micrograms/ml in an undefined growth nutrient or fatty acid-deficient nutrient supplemented only with palmitic acid. When A. laidlawii is propagated in fatty acid-deficient nutrient supplemented with linoleic acid, however, the organisms become 40 times more sensitive to the growth inhibitory effect of this antibiotic. The actual fatty acid composition of the membranes would indicate that a higher degree of unsaturation enhances ionophore activity.

Factors affecting DNA sequence selectivity of nogalamycin intercalation: the crystal structure of d(TGTACA)2-nogalamycin2.

As part of an investigation into the sequence selectivity of the nogalamycin-DNA interaction, the 1.58 A structure of nogalamycin complexed with d5'(TGTACA)2 has been determined by single-crystal X-ray analysis. The complex crystallised in the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 26.3 A, b = 52.0 A and c = 67.1 A, incorporating two B-DNA duplexes and four nogalamycin molecules in the asymmetric unit. The final refined structure included 97 water molecules, one spermine molecule, two acetate ions and one sodium ion, yielding an overall R factor of 19.2% (calculated using all 12,358 reflections in the resolution range 10.7 to 1.6 A) and an Rtree of 23.7% (using 1229 test reflections). The d5'(TGTACA)2 sequence was designed to include the d5'(TpG) pyrimidine-purine base step that has been ascertained as a preferential intercalation site. The complexes in the asymmetric unit are globally similar; one nogalamycin molecule intercalates between each d5'(TpG) step in each duplex. The DNA of each complex exists as a distorted B-DNA duplex displaying some Z-DNA character in the form of C3' endo sugars at some residues. Structural comparisons between the d5'(TGTACA)2-nogalamycin2 complex and the complexes of this drug with the sequences d5'(TGATCA)2 and d5'(5MeCGT(pS)A5MeCG)2 highlight differences in binding interactions between nogalamycin and these various triplet DNA binding sites, with regards to the stability of drug intercalation, which in turn is correlated to effective levels of cytotoxicity towards tumour cells. The number of both direct and water-mediated hydrogen bonds and van der Waal's interactions between substituents of nogalamycin and the d5'(TGTACA)2 and d5'(5MeCGT(pS)A5MeCG)2 sequences are significantly greater than those made with the d5'(TGATCA)2 sequence, suggesting that the central d5'(TpA) in the former confers additional stability to the complex once the drug has bound.

Surface point mutations that significantly alter the structure and stability of a protein's denatured state.

Significantly different m values (1.9-2.7 kcal mol-1 M-1) were observed for point mutations at a single, solvent-exposed site (T53) in a variant of the B1 domain of streptococcal Protein G using guanidine hydrochloride (GuHCl) as a denaturant. This report focuses on elucidating the energetic and structural implications of these m-value differences in two Protein G mutants, containing Ala and Thr at position 53. These two proteins are representative of the high (m+) and low (m-) m-value mutants studied. Differential scanning calorimetry revealed no evidence of equilibrium intermediates. A comparison of GuHCl denaturation monitored by fluorescence and circular dichroism showed that secondary and tertiary structure denatured concomitantly. The rates of folding (286 S-1 for the m+ mutant and 952 S-1 for the m- mutant) and the rates of unfolding (11 S-1 for m+ mutant and 3 S-1 for the m- mutant) were significantly different, as determined by stopped-flow fluorescence. The relative solvation free energies of the transition states were identical for the two proteins (alpha ++ = 0.3). Small-angle X-ray scattering showed that the radius of gyration of the denatured state (Rgd) of the m+ mutant did not change with increasing denaturant concentrations (Rgd approximately 23 A); whereas, the Rgd of the m- mutant increased from approximately 17 A to 23 A with increasing denaturant concentration. The results indicate that the mutations exert significant effects in both the native and GuHCl-induced denatured state of these two proteins.

Cloning and characterization of a novel histone acetyltransferase homologue from the protozoan parasite Toxoplasma gondii reveals a distinct GCN5 family member.

In an effort to identify gene products involved in transcriptional regulation in apicomplexan parasites, the Toxoplasma gondii expressed sequence tag (EST) database was examined for sequences containing similarity to known transcriptional components. One EST (dbEST ID #466792) exhibited strong similarity to yeast GCN5 and other histone acetyltransferases (HATs). Primers were designed based on the EST sequence and used to amplify an 850 bp fragment (containing an intron) from T. gondii genomic DNA which was used to identify four cDNA clones from a tachyzoite cDNA library. The complete open reading frame (ORF) of 3.5 kb was elucidated using 5' RACE and genomic sequence. The deduced amino acid sequence of the coding region shows that the C-terminal domain possesses unequivocal similarity to GCN5 family members. However, unlike other lower eukaryotes, T. gondii GCN5 has an extended N-terminal domain similar in length, but not in composition, to metazoan HAT proteins. These features distinguish T. gondii GCN5 as a novel member of the GCN5 family. A portion of the cDNA sequence was used as a probe to isolate three overlapping clones from a T. gondii genomic library, generating a approximately 7.5 kb map of the GCN5 locus which contains seven exons separated by six introns. Southern analysis verifies the predicted map and suggests that a similar locus may be present elsewhere in the genome.

Identification and characterization of a target antigen of a monoclonal antibody directed against Eimeria tenella merozoites.

Monoclonal antibodies (Mab) were produced against Eimeria tenella merozoites. A single Mab, LPMC-61, was selected because of its ability to bind to merozoites by indirect immunofluorescence assay (IFA) and to inhibit in vitro sporozoite development. Mab LPMC-61 reacts with an approx. 10-12-kDa merozoite polypeptide in reduced SDS-PAGE, but with an approx. 80-kDa protein in non-reduced SDS-PAGE. The monoclonal recognizes similarly sized polypeptides in E. tenella sporozoites, oocysts and schizonts. A partial cDNA (LPMC-61f) encoding the LPMC-61 antigen was identified from an E. tenella sporozoite cDNA library in bacteriophage lambda gt11. In addition to Mab LPMC-61, the recombinant beta-galactosidase/LPMC-61f fusion protein is recognized by hyperimmune rabbit anti-E. tenella sporozoite serum, rabbit anti-E. tenella merozoite serum, and E. tenella-infected and immune chicken sera. DNA sequencing of LPMC-61f cDNA showed that the putative protein has an unusual tandem, non-perfect repeated sequence, with glutamine comprising about 48% of the predicted amino acids. A hydropathicity plot of the predicted amino acid sequence shows a central hydrophilic region, consisting of the repeated sequences, surrounded by hydrophobic regions on both sides. Since the merozoite stage of avian Eimeria has been implicated in the induction of a protective immune response in chickens, LPMC-61 may be an important immunogen for use as a vaccine against E. tenella.

Synthesis and endectocidal activity of novel 1-(arylsulfonyl)-1-[(trifluoromethyl)sulfonyl]methane derivatives.

We have recently synthesized a series of novel disulfonylmethane compounds that have shown anthelmintic and insecticidal (endectocidal) activity. Several analogues have shown activity against the internal nematode Haemonchus contortus. In sheep studies, these analogues have shown 100% control of this internal parasite at a 10 mg/kg rate. In vitro activity against the biting flies, Stomoxys calcitrans and Haematobia irritans, has been observed at rates as low as 25 and 2.3 ppm, respectively. Only marginal activity against the liver fluke Fasciola hepatica and Trichostrongylus colubriformis was seen. Respiratory control index values on rat liver mitochondria for this series suggested uncoupling of oxidative phosphorylation as a mechanism of action. Compound 1 is considered to be a promising agent for treatment of parasitized sheep.

Use of internal controls to increase quantitative capabilities of the ribonuclease protection assay.

Through the use of two internal controls, we have developed an improved method of quantitating ribonuclease protection assay (RPA) results. A truncated sense RNA fragment and an antisense RNA fragment for the gene of interest were transcribed from PCR fragments containing T7 bacterial promoters. An 18S ribosomal RNA fragment was also used. When radiolabeled antisense and 18S probes, along with sense fragment and sample RNA, were hybridized, digested with RNase A/T1 and gel-electrophoresed, three distinct bands resulted. The antisense RNA fragment bound to the sense RNA fragment confirmed the integrity of the reaction. The antisense RNA fragment bound to endogenous mRNA measured the amount of specific gene expression in the sample. The 18S RNA fragment bound to endogenous mRNA determined the actual amount of sample added to the gel. Using the specific activities of the antisense and 18S transcripts, and scintillation counts of the protected fragments, we calculated the amounts of message and total RNA on the gel, determining picogram of message per microgram of total RNA. Final results were not based on assumed original amounts of RNA placed in the assay nor were they biased by lane-to-lane variations. Through the described adaptations, we have developed a well-controlled RPA that accurately and reproducibly quantifies gene expression.

Increased responsiveness to intravenous lipopolysaccharide challenge in steers grazing endophyte-infected tall fescue compared with steers grazing endophyte-free tall fescue.

Fescue toxicosis in cattle occurs as a result of consumption of ergot alkaloids in endophyte-infected (E+, Neotyphodium coenophialum) tall fescue (Festuca arundinacea). The condition is characterized by pyrexia, decreased weight gains, rough hair coats, and decreased calving rates. The objective of this experiment was to investigate whether steers grazing E+ fescue have altered host response to lipopolysaccharide (endotoxin, LPS) challenge compared with steers grazing endophyte-free (E-) fescue. Angus steers (n=8) had continuously grazed either E+ (n=4) or E- (n=4) tall fescue grass for 8 months prior to the experiment. The E+ steers had lower body weight, depressed average daily gain, and decreased basal serum prolactin compared with the E- steers prior to LPS administration. Each steer received a single bolus i.v. injection of LPS (0.2 microgram/kg body weight; Escherichia coli; 026:B6) dissolved in sterile saline, and blood was serially collected every 30 min for 4 h and at 24 h post LPS administration. LPS increased serum tumor necrosis factor-alpha (TNF-alpha), cortisol, and haptoglobin but decreased plasma glucose and IGF-I. Importantly, however, TNF-alpha, cortisol, and IGF-I responses to LPS were greater in E+ compared with E- steers. These results indicated that animals grazing E+ fescue had altered integrated metabolic host response compared with animals grazing E- fescue. Potentially, combined exposure to E+ fescue and a bacterial LPS could have greater deleterious effects on the animal compared with exposure to only one of the two and would likely lead to increased catabolism.

Cold acclimation of obese (ob/ob) mice: effects of energy balance.

Obese (ob/ob) and lean mice at 4 weeks of age were housed at 23 degrees C or 14 degrees C for 4 to 8 weeks to examine effects of acclimation to mild cold on energy balance. Energy intake of young lean mice increased by about 50% when housed at 14 degrees C, but energy intake of cold-acclimated obese mice increased by only 8%. Efficiency of energy retention (ratio of energy gain to energy intake) in obese mice declined from 22% +/- 1.2% at 23 degrees C to 10% +/- 1.8% after 4 weeks at 14 degrees C. Lean mice exhibited a less pronounced response to temperature; their efficiency of energy retention declined from 7% +/- 1.3% at 23 degrees C to 4% +/- 2.2% after 4 weeks at 14 degrees C. After 8 weeks of cold exposure, body weights and efficiency of energy retention became equal in obese and lean mice. Calculated heat production of cold-acclimated obese and lean mice was 40% higher than that of respective controls. Obese mice reacclimated to 23 degrees C after being kept for 4 weeks at 14 degrees C consumed the same amount of energy and were 16% more efficient than obese maintained at 23 degrees C; reacclimated lean mice consumed 12% more energy but were 53% less efficient than lean mice maintained at 23 degrees C. The results indicate that obese mice are able to increase heat production and markedly reduce their efficiency of energy retention when acclimated to mild cold but that they, unlike lean mice, rapidly revert to a high efficiency of energy retention after 4 weeks of reacclimation to 23 degrees C.

Cold acclimation of obese (ob/ob) mice: effects on skeletal muscle and bone.

Obese (ob/ob) and lean mice at 4 weeks of age were housed at 23 degrees C or 14 degrees C for 4 or 8 weeks to determine effects of acclimation to mild cold on the growth of skeletal muscle, bone, and fat. Body weights at 12 weeks of age averaged 48 +/- 0.6 g and 27 +/- 1.9 g for obese mice housed at 23 degrees C and 14 degrees C and 29 +/- 0.5 g and 26 +/- 0.6 g and 26 +/- 0.6 g for lean mice housed at 23 degrees C and 14 degrees C, respectively. At 23 degrees C, muscle weights of obese mice were approximately 60% of those in lean mice. Muscles of obese mice did not grow during the first 4 weeks at 14 degrees C (4 to 8 weeks of age) but did show a small gain during the second 4 weeks (9 to 12 weeks of age) at 14 degrees C. As a result, by the end of 8 weeks at 14 degrees C, muscles of obese mice weighed only 35% to 45% as much as muscles of lean mice. Growth of the tibia and femur followed the same pattern as the muscles. Obese mice housed at 23 degrees C from 4 to 12 weeks of age contained about six times as much fat as lean mice at this age. Although exposure to 14 degrees C for 8 weeks depressed the accumulation of fat in obese mice, they still contained approximately three times the percentage body fat as lean counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovarian steroids modulate content and secretion of immunoreactive gonadotropin-releasing hormone in a rabbit endometrial cell line.

Incubation of a rabbit endometrial cell line (HRE-H9 cells) with KCl (5-60 mM) for 30 min enhanced IR-GnRH secretion, with 30 and 60 mM producing the greatest stimulatory effect (280 +/- 19% and 298 +/- 49% of control group, respectively). By adding 30 mM KCl into HRE-H9 culture and increasing the incubation time to 90 min, there was a stepwise increase in IR-GnRH secretion. In the third experiment, treatment of HRE-H9 cells with estradiol (E2, 10(-9)-10(-8) M) for 48 h stimulated IR-GnRH secretion (215 +/- 17%, 168 +/- 19%, respectively), whereas P4 treatment did not produce any significant change. Treatment with E2 + P4 at all doses tested (10(-10)-10(-6) M) augmented the secretion of IR-GnRH (140 +/- 16%, 153 +/- 14%, 276 +/- 23%, 259 +/- 26%, 198 +/- 16%, respectively). Increased IR-GnRH secretion by E2 (10(-9) M) and E2 + P4 (10(-8)-10(-7) M) resulted in a reduction in cell content of IR-GnRH (p < 0.05). In conclusion, secretion of IR-GnRH by HRE-H9 cells can be induced by KCl depolarization. Treatment of HRE-H9 cells with E2 and E2 + P4 enhanced their secretion of IR-GnRH. Under such conditions, secreted IR-GnRH appears to be derived primarily from intracellular IR-GnRH pools.

Quantification of rosuvastatin in human plasma by automated solid-phase extraction using tandem mass spectrometric detection.

An assay employing automated solid-phase extraction (SPE) followed by high-performance liquid chromatography with positive ion TurboIonspray tandem mass spectrometry (LC-MS-MS) was developed and validated for the quantification of rosuvastatin (Crestor) in human plasma. Rosuvastatin is a hydroxy-methyl glutaryl coenzyme A reductase inhibitor currently under development by AstraZeneca. The standard curve range in human plasma was 0.1-30 ng/ml with a lower limit of quantification (LLOQ) verified at 0.1 ng/ml. Inaccuracy was less than 8% and imprecision less than +/-15% at all concentration levels. There was no interference from endogenous substances. The analyte was stable in human plasma following three freeze/thaw cycles and for up to 6 months following storage at both -20 and -70 degrees C. The assay was successfully applied to the analysis of rosuvastatin in human plasma samples derived from clinical trials, allowing the pharmacokinetics of the compound to be determined.

Prey size-distributions and size-specific foraging success of Ambystoma larvae.

We examined how prey size-distributions influence size-specific foraing rate and food gain, i.e., food intake scaled to metabolic demands, in Jefferson's and small-mouth salamander larvae. Ambystoma jeffersonianum larvae sampled on 17 dates from a farm pond whose fauna was dominated by macrozooplankton and chironomid larvae were rarely gape-limited, and total volume of food in the stomach (VS) showed only a slight tendency to increase with larval size. Although 15 of 17 correlation coefficients of VS with larval size were positive, only 1 of 17 correlations were statistically significant, and body size explained only 8% of the overall variation in VS. Correlation coefficients of food gain and body size were positive in 9 cases and negative in 8, but only 3 were statistically significant.In contrast, Ambystoma texanum larvae in 42 samples taken from five sites dominated by macrozooplankton as well as relatively large isopods and amphipods were almost always gape-limited, and VS tended to increase markedly with larval size. 40 of 42 correlation coefficients of VS and larval size were positive, and 19 correlations were statistically significant. Body size in turn explained about 35% of the overall variation in VS. Correlation coefficients of food gain and larval size were positive in 32 of 42 samples, and 9 of 10 significant correlations were positive.When food is limiting and prey selection is not limited by gape, smaller larvae may grow as fast or in some cases faster than larger larvae because they are nearly as effective foragers, but have lower metabolic demands. Larger larvae may in turn grow faster than smaller larvae in environments which support a broad size spectrum of prey, particularly when gape limitations are highly disproportionate among size classes. The growth rate of larvae in one size class relative to another depends primarily on the extent to which increased foraging rate compensates for higher energy demands as body size increases. Size-specific foraging rate may in turn be strongly influenced by the prey size-distribution within a habitat. These relationships suggest that relative size is not always a good a priori predictor of exploitative competitive ability.

Life change and onset of cancer in identical twins.

The relationship of life change to the onset of cancer was studied in 22 pairs of HLA-identical siblings who were discordant for hematologic malignancies. The twin pairs were hospitalized for bone marrow transplantation. Life change was measured using a well-validated instrument, the Schedule of Recent Experiences (SRE). Contrary to our expectations, we were unable to document increased life changes in the sick twins. The timing of administration of the SRE with respect to the transplant did influence reporting of life events. However, regardless of timing of administration, in the period antedating the diagnosis of malignancy the healthy donor twins had increased or equivalent life changes when compared to their sick twins.

A randomised clinical trial of nicotine patches for treatment of spit tobacco addiction among adolescents.

BACKGROUND: This study tested the efficacy of nicotine patches in combination with behavioural therapy for the treatment of adolescent spit tobacco addiction. Prior interventions had resulted in mean cessation rates below 15% at one year. METHODS: This study, the PATCH Project, used a three group, placebo controlled, randomised clinical trial design. The control group received a standard 3-5 minute counselling followed by a two week follow up phone call. The two intervention groups received a six week behavioural intervention; in addition, one group received active nicotine patches while the other group received placebo patches. Both groups received quarterly stage based telephone counselling. RESULTS: At one year, the usual care group's spit tobacco cessation rate was 11.4% (exact 95% confidence interval (CI) 6.1% to 19.1%), placebo patch 25.0% (95% CI 16.9% to 34.7%), and the active patch 17.3% (95% CI 10.4% to 26.3%). When both patch groups were combined, the cessation rate was 21.2% (95% CI 15.7% to 27.6%). The cessation rates for active and placebo patch were not significantly different (exact two sided p = 0.22), while the combined patch groups had a significantly greater cessation rate than usual care (exact two sided p = 0.04). CONCLUSIONS: The behavioural intervention proved to be about twice as successful as previous interventions, but the nicotine patch offered no improvement in cessation rates. The behavioural intervention is based on publicly available materials and can be easily adapted for widespread use, particularly in high schools.

Monosaccharide transport by Eimeria tenella sporozoites.

Eimeria tenella sporozoites were incubated in the presence of 3 different [14C]-labeled sugars; D-glucose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose. The initial velocity, Vi, of uptake of D-glucose and 2-deoxy-D-glucose was similar, 41 micrograms/10(10) sporozoites/min and 46 micrograms/10(10) sporozoites/min, respectively; whereas that for 3-O-methyl-D-glucose was significantly lower, 17 micrograms/10(10) sporozoites/min. Initial velocity studies also revealed that glucose uptake was a saturable event, with an apparent KT of 20 mM and an apparent Vmax of 312 micrograms/10(10) sporozoites/min. Uptake was unaffected by exogenous sodium levels or the presence of ouabain. However, 0.1 mM phloretin significantly inhibited glucose uptake. Thus, it would appear that E. tenella sporozoites possess a Na-independent, phloretin-sensitive, carrier-mediated monosaccharide-transport system.

Influence of monensin on cation influx and glycolysis of Eimeria tenella sporozoites in vitro.

Extracellular Eimeria tenella sporozoites exposed to 1.0 microgram/ml monensin at 40 C had an accelerated rate of sodium influx as well as an increased rubidium uptake that was inhibited by the cardiac glycoside, ouabain. These results suggested the presence of a functional (Na+-K+)-ATPase and its stimulation by monensin. Under the same conditions, sporozoite ATP concentrations declined, lactate production increased and the rate of amylopectin utilization was enhanced. Exposure to monensin also appeared to stimulate the rate of sporozoite glycolysis. The results of this study demonstrated that the cidal effect of monensin on extracellular sporozoites was caused by the capability of the ionophore to act as a transmembrane sodium carrier.